We obtained genetic information from specimens that were collected in São Paulo from March 1995 to October 1997 (Table). During 1995 and 1996, relatively few measles cases were reported in São Paulo; six acute-phase serum samples were obtained from confirmed, sporadic cases that occurred as early as 2 years before the outbreak. In 1997, a resurgence of measles occurred in São Paulo, beginning in the fall and peaking in late winter (Figure 1). Twenty-three samples were obtained from cases that occurred during the first 42 weeks of 1997; 11 of these samples were obtained in August 1997 during the peak of the outbreak (Table). We obtained most of the outbreak specimens from young adults, which is consistent with the observation that most of the measles cases occurred in unvaccinated, young adults (6). Measles-specific immunoglobulin (Ig) M responses were detected in all the patients listed in the Table, except one. The serum sample from case number 802 was negative, probably because it was obtained <3 days after rash onset. However, this case was confirmed by a positive virus isolation (Table).

Virus isolation was not attempted for the serum samples, but five virus isolates were obtained from the PBMC specimens (Table) obtained during 1997. The sequences coding for the carboxyl terminus of the nucleoprotein (456 nucleotides) of these isolates were compared with the sequences of the WHO reference strains (12). The results indicated that these Brazilian viruses all had identical N gene sequences and were members of genotype D6 (Figure 2). The N gene sequence of MVi/SãoPaulo.BRA/42.97/35175 has been deposited in GenBank (accession no. AF495863) as a reference sequence for this outbreak. The complete sequence of the H gene was also obtained for MVi/SãoPaulo.BRA/42.97/35175. Comparison of this H gene sequence with the sequences of the H genes from the WHO reference strains confirmed the placement of these viruses in genotype D6 (data not shown).

For the rest of the specimens from 1997, nucleotide sequences were obtained from PCR products that were amplified directly from RNA extracted from the PBMC specimens. In most cases, we had to perform a nested PCR reaction to get sufficient material for sequencing. All specimens obtained in 1997, either just before or during the peak of the outbreak, were placed in genotype D6. Most of the N gene sequences obtained from these specimens were identical to each other and to the N gene sequences obtained from the five isolates from 1997. Only three of the specimens had sequences that differed from the majority sequence by a single nucleotide.

We were also able to amplify measles-specific PCR products from the serum samples obtained from sporadic measles cases that occurred in 1995 and 1996. Two of these specimens had nucleotide sequences in genotype A, which contains all vaccine viruses. Both specimens were obtained from 9-month-old infants who had received their measles vaccination 12–13 days before collection of the serum specimen. One of the samples obtained in 1995 had an N gene sequence in genotype D5, while the other three serum samples had sequences in genotype D6 identical to the D6 sequences obtained during the outbreak. These data showed that D6 viruses and wild-type viruses from genotypes other than D6 were present in São Paulo as early as 2 years before the peak of the outbreak in 1997.

We compared the N gene sequences from the genotype D6 viruses in the São Paulo cases with N gene sequences from genotype D6 viruses isolated in other parts of South America (20–24) and the world (Figure 2). For simplicity, three representative sequences from the South American viruses are shown in the phylogenetic tree. The sequences from the São Paulo outbreak were identical to those obtained from viruses isolated in Brazil (i.e.,Valenca, Rio de Janeiro, Belo Horizonte, Angra do Reis, and Curitiba) (20,21), Argentina (22,23), Uruguay (24), and Bolivia in 1997 and 1998. This homogenous group also included two viruses that were imported into the United States from Brazil in 1997 (25). The sequences from genotype D6 viruses imported into the United States (26, unpub. data) from European sources and genotype D6 viruses isolated in Europe (27,28) were slightly different from the South American D6 group. We found that the sequences from the South American viruses were most closely related to a genotype D6 virus isolated in Luxembourg in 1997, although no foreign source was identified for the outbreak in Brazil. Overall, the N genes sequences of D6 viruses within the South American group differed by no more than 0.4% but differed from other genotype D6 sequences by as much as 1.4%.

This report presents the virologic surveillance data from the measles outbreak that occurred in São Paulo, Brazil, during 1997. Sequence data from the specimens obtained during the outbreak indicated the presence of measles viruses belonging to genotype D6, which is one of the prevalent genotypes of measles virus in Europe. Frequently associated with imported measles cases from European countries (8,26), D6 viruses have been isolated in the United Kingdom, Spain, Germany, Russia, Poland, Denmark, and Luxembourg (27–31). During 1997–2000, these genotype D6 viruses were imported into the United States from Italy, Greece, Ukraine, Croatia, Cyprus, and the United Kingdom, and from the large measles outbreak that occurred in São Paulo (Rota, unpub. data). However, because neither conventional case nor virologic surveillance was conducted in Brazil before the start of the measles control program in 1991, it is not possible to determine if the D6 viruses were recently introduced into Brazil by importation or if they represent continued circulation of viruses indigenous to Brazil. Our data show that D6 viruses indistinguishable from the D6 viruses isolated during the São Paulo outbreak were responsible for sporadic measles cases in São Paulo for at least 2 years before the outbreak. In a large urban area such as São Paulo, the virus will likely be continually present either from low-level circulation or frequent importation. In late 1997, a change occurred in the epidemiologic circumstances, resulting in a large outbreak of the measles virus. Factors that contributed to the outbreak included the failure to conduct a timely follow-up vaccination campaign in 1995 and the low level of vaccination coverage among infants achieved by routine health services. Additionally, a large number of unvaccinated young adults who had escaped measles infection had moved to São Paulo from rural areas. The high population density in São Paulo also facilitated rapid spread of the virus (7).

Laboratory-based surveillance for measles was established in Brazil in the early 1990s. Although virologic surveillance was certainly incomplete, some information is available about measles genotypes present in Brazil before the 1997 outbreak in São Paulo. Genotype D5 was detected in a sporadic case in São Paulo in 1995 and in sporadic cases that occurred in the state of Bahia in 1996 (21). Genotype D5 viruses that circulate in Japan have been associated with importation of viruses from Japan into other countries (26,32,33). The source of the genotype D5 viruses detected in São Paulo and Bahia was not identified. Genotype C2 viruses were detected in a small outbreak in the state of Santa Catarina in 1996 (21). C2, another indigenous genotype in Europe, is often associated with imported cases in other parts of the world (27–31).

Fever and rash occur in approximately 5% of measles vaccine recipients (34). In areas where wild-type virus is circulating, vaccine reactions may be classified as measles cases. Distinguishing a vaccine reaction from a measles case is not possible with the currently available serologic methods. Only the genetic analysis of virus isolates can confirm the presence of either a vaccine or wild-type virus. In this study, two vaccine viruses were detected in persons who had received vaccine 12 and 13 days before collection of specimens. In vaccine reactions, rash and fever typically occur 7 to 12 days after vaccination; the virus can be detected in circulating lymphocytes even after the rash and fever have resolved.

The situation in Brazil is now very similar to that in the United States in the early 1990s. From 1989 to 1991, the United States experienced a resurgence of measles, with >50,000 reported cases. Virologic surveillance suggested that virus from a single genotype, D3, had seeded the entire country. As was the case with the D6 viruses from South America, the D3 viruses from many different areas in the United States had nearly identical sequences (8). Therefore, we conclude that when measles outbreaks occur in areas that have good measles control, genetic analysis of viruses will confirm the presence of a single chain of transmission. In contrast, in areas where measles control programs are less advanced, genetic studies have identified multiple chains of transmission within a genotype (17).

Initiation of a two-dose vaccination schedule, along with the success of the PAHO strategy in the Americas, resulted in historically low numbers of cases in the United States in the years following the resurgence there; both standard epidemiologic surveillance and molecular epidemiologic surveillance have documented the sustained interruption of transmission of the genotype D3 viruses (1,2,8,35–37). Similarly, the pattern of viral genotypes found in Brazil and other parts of South and Central America will help to assess the success of the PAHO measles control programs. Genotype D6 viruses were circulating in Haiti and the Dominican Republic during 2001 but have not been isolated from ongoing chains of transmission in any other countries in the region (36). During 2002, virologic surveillance showed that measles cases imported into El Salvador from Europe were genotype D7 (38). As the number of measles cases continues to decrease in the Americas, increasing virologic surveillance activities will be important. This virologic surveillance will document the interruption of indigenous transmission of the genotype D6 viruses in Brazil and in other parts of South America.